Abstract
BackgroundNon-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. NTHi is known to form biofilms. Resistance of bacterial biofilms to clearance by host defense mechanisms and antibiotic treatments is well-established. In the current study, we used stable isotope labeling by amino acids in cell culture (SILAC) to compare the proteomic profiles of NTHi biofilm and planktonic organisms. Duplicate continuous-flow growth chambers containing defined media with either “light” (L) isoleucine or “heavy” (H) 13C6-labeled isoleucine were used to grow planktonic (L) and biofilm (H) samples, respectively. Bacteria were removed from the chambers, mixed based on weight, and protein extracts were generated. Liquid chromatography-mass spectrometry (LC-MS) was performed on the tryptic peptides and 814 unique proteins were identified with 99% confidence.ResultsComparisons of the NTHi biofilm to planktonic samples demonstrated that 127 proteins showed differential expression with p-values ≤0.05. Pathway analysis demonstrated that proteins involved in energy metabolism, protein synthesis, and purine, pyrimidine, nucleoside, and nucleotide processes showed a general trend of downregulation in the biofilm compared to planktonic organisms. Conversely, proteins involved in transcription, DNA metabolism, and fatty acid and phospholipid metabolism showed a general trend of upregulation under biofilm conditions. Selected reaction monitoring (SRM)-MS was used to validate a subset of these proteins; among these were aerobic respiration control protein ArcA, NAD nucleotidase and heme-binding protein A.ConclusionsThe present proteomic study indicates that the NTHi biofilm exists in a semi-dormant state with decreased energy metabolism and protein synthesis yet is still capable of managing oxidative stress and in acquiring necessary cofactors important for biofilm survival.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0329-9) contains supplementary material, which is available to authorized users.
Highlights
Non-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis
Whether HbpA’s primary role is binding heme, binding glutathione, or perhaps both, it is interesting to hypothesize that the promiscuity of HbpA’s substrate binding may be by necessity or design and could play an important role in H. influenzae biofilm growth and virulence. It is clear from the current study, as well as previous studies, that some common factors are important in biofilm formation
H. influenzae within a biofilm need ways of dealing with the various effects of oxidative stress and damage
Summary
Non-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative organism that is a typical component of the normal human nasopharyngeal flora. Under certain conditions, this bacterium can be an opportunistic pathogen causing upper and lower respiratory tract infections such. H. influenzae is able to form biofilms under both in vitro and in vivo conditions [6,7,8,9,10,11,12] These biofilms are bacterial communities that exhibit characteristics which differentiate them from planktonic organisms [13]. This matrix most likely plays a role in resistance to host defenses and antimicrobial therapies
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